Bacterial shape is determined and maintained by an extracellular, rigid structure composed of peptidoglycan, a polymer of repeated units of a disaccharide peptide monomer. The essential enzymes that synthesize these monomers and assemble them into polymers are well characterized and are the targets of many important antibiotics. However, the mechanism by which the cytosolically synthesized monomers are translocated across the cytoplasmic membrane to their site of polymerization remains a fundamental unresolved issue. We propose to investigate translocation during the non-essential process of sporulation in the Gram-positive bacterium Bacillus subtilis. The endospore contains a thick layer of peptidoglycan, the spore cortex, which is largely responsible for its heat and desiccation resistance. One gene required for spore cortex formation is a sporulation-specific and therefore non-essential homolog of two essential genes that encode integral membrane proteins found in all bacteria with a cell wall. Mutations in these genes lead to defects in cell shape consistent with a proposed role in cell wall synthesis. The objectives of this project are: (1) to test the hypothesis that this protein mediates translocation of the peptidoglycan precursor during sporulation; (2) to characterize the multiprotein complexes including the protein that mediate cell wall synthesis; and (3) to examine the subcellular localization of this protein and other proteins involved in spore peptidoglycan synthesis and analyze the underlying mechanisms responsible for their targeting. The increasing prevalence of strains of antibiotic-resistant bacteria emphasizes the importance of identifying potential new antibiotic targets and the protein(s) that mediate precursor translocation during peptidoglycan synthesis are excellent candidates. Relevance to Public Health Bacterial infections are usually treated successfully with antibiotics, drugs that interfere with the ability of the bacteria to grow and multiply. However, an increasing number of bacteria are resistant to these drugs. The aim of this work is to identify aspects of the physiology of the bacteria that might become the targets of new antibiotics. PUBLIC HEALTH RELEVANCE We will study the process by which bacteria construct their cell wall, the structure that determines and maintains their shape. Although this process is the target of many presently used antibiotics including penicillin, bacteria are quickly becoming resistant to these drugs. We expect that new understanding of this process will lead to the development of new antibiotics.